Nucleotide sequences for the control of the expression of DNA sequences in a cell host

ABSTRACT

The present invention provides nucleotide sequences from Bacillus bacteria, which control the expression of other DNA sequences in a cell host.

[0001] The object of the invention is nucleotide sequences of bacteria,in particular Gram⁺ bacteria such as bacteria of the Bacillus type andmore particularly nucleotide sequence of the cryIIIA gene for thecontrol of the expression of DNA sequences in a cell host.

[0002] The cryIIIA gene codes for a toxin specific for the Coleopteraand is weakly expressed by Bacillus thuringiensis when it is cloned in alow copy number plasmid.

[0003]Bacillus thuringiensis is a Gram-positive bacterium which producessignificant quantities of proteins in the form of crystals having atoxic activity towards insect larvae. Two groups of crystal proteins areknown, based on the amino acid sequences and the toxicity specificities:

[0004] 1) the class of the Cry toxins (I, II, III, etc. . . . ) whichhave similar structures;

[0005] 2) the class of the Cyt toxins, which is not related to the Cryclass (Höfte, H et al. 1989, Microbiol. Rev. 53: 242-255)

[0006] These toxins of B. thuringiensis are of general interest for thepurpose of the development of bio-pesticides and also in as much as thesynthesis of crystal proteins is known to be perfectly coordinated withthe sporulation phase of the organism, making this organism interestingfor the study of genetic regulation in sporulating Gram-positivebacteria.

[0007] Various mechanisms implicated in the regulation of the synthesisof the crystal proteins of B. thuringiensis have been described. Thehigh level of expression of these proteins is attributed, at least inpart, to the stability of the mRNA. Some authors have attributed thestability of this mRNA to the presence downstream from the gene for thetoxin of a structure playing a terminator role which might act as apositive retro-regulator by protecting the 3′ end of the mRNA fromdegradation by nucleases, thus increasing the half-life of thetranscripts (Wong, H. C. et al., 1986 Proc. Natl. Acad. Sci. USA 83:3233-3237).

[0008] A hypothesis has also been put forward concerning the presence ofpolypeptides implicated in the synthesis of crystal proteins,polypeptides which are supposed to act either by directing the foldingof the protein in the form of a protein having a stable conformation orto protect these proteins from proteolytic degradation.

[0009] Studies with the electron microscope and biochemical studies ofsporulation in B. thuringiensis show that the production of the crystalprotein is dependent on sporulation and is located in the mother cellcompartment (Ribier, J. et al. 1973 Ann. Inst. Pasteur 124A: 311-344).

[0010] Recently, two sigma factors, sigma 35 and sigma 28, whichspecifically direct the transcription of the cryIA genes have beenisolated and characterized. These amino acid sequences exhibit anidentity of 88 and 85% with the sigma factors E and K of Bacillussubtilis, respectively (Adams, L. F., 1991, J. Bacteriol. 173:3846-3854). These sigma factors are produced exclusively in sporulatingcells and are capable of functioning in the mother cell compartment,confirming that the expression of the genes for the crystal protein iscontrolled in time and space. Thus, in the prior art it has beenconcluded that the expression of the gene with time is, at least inpart, ensured by the successive activation of the sigma factors specificfor sporulation. Hitherto, three groups of promoters have beenidentified. Two of these groups include promoters recognized by specificsigma factors and, according to the prior art, the sigma factorsassociated with the third group if promoters (including that of thecryIIIA gene) have not been identified (Lereclus, D., et al. 1989American Society for Microbiology, Washington, D.C.).

[0011] Finally, the copy number of the plasmid bearing the gene seems tobe an important factor for the expression of the cry gene in B.thuringiensis. In the B. thuringiensis wild type strain, the cry genesare localized on large plasmids, present in a low number of copies.

[0012] Cloning experiments with a 3 kb HindIII fragment cloned in a lowcopy number plasmid lead to a low production of toxins in anon-crystal-forming strain (cry⁻) of B. thuringiensis. On the otherhand, large quantities of toxins are synthesized when the gene is clonedin plasmids of high copy number (Arantes, O et al. 1991, Gene 108:115-119).

SUMMARY OF THE INVENTION

[0013] The object of the invention is agents making it possible toobtain a high level of expression of the protein encoded in the cryIIIAgene and more generally agents making it possible to control the levelof expression of DNA sequences coding for a specific protein of interestin bacterial strains, preferably Gram⁺ strains such as Bacillus strains,since it is possible to obtain this expression when the coding DNAsequence is located on a vector, in particular on a plasmid of low copynumber.

[0014] Generally speaking, the invention relates to an expression systemcomprising a DNA sequence, able to intervene in the control of theexpression of a coding nucleotide sequence and obtained by associatingtwo distinct nucleotide sequences intervening in different but,preferably, not dissociable ways in the control of the expression of thecoding sequence. The first nucleotide sequence exhibits a promoteractivity whereas the second sequence, initiated by the promoter activityof the first, intervenes to enhance the expression of the gene. The DNAsequence of the invention makes it possible to attain a high level ofexpression of the coding part of a gene in a bacterium, in particular aGram⁺ type of bacterium.

[0015] The first nucleotide sequence of the expression system of thepresent invention identified in the framework of the present demand asbeing the promoter consists of either the promoter of the host strain inwhich the gene of interest to be expressed is introduced, or of anexogenous promoter, functional in the host used. The second nucleotidesequence of the expression system of the invention identified in thepresent application as being the “downstream region” designates anysequence preferably situated between the promoter and the sequencecoding for a gene to be expressed, able to play a role particularly atthe post-transcriptional level when the gene is expressed. Moreparticularly, the downstream region does not act directly on thetranslation of the coding sequence to be expressed.

[0016] In a preferred manner, the “downstream region” consists of anucleotide sequence, particularly an S2 sequence or a sequence analogousto S2, containing a region essentially complementary to the 3′ end ofthe RNA, particularly the 16S RNA, of the ribosomes of bacteria,particularly of Gram⁺ bacteria of the Bacillus type.

[0017] The nucleotides forming the DNA sequence according to theinvention may or may not be consecutive in the sequence from which theDNA sequence is defined.

[0018] In the context of the present application the expression “DNAsequence able to intervene in the control of the expression of a codingnucleotide sequence” expresses the capacity of this DNA sequence toinitiate or prevent the expression of the coding sequence or to regulatethis expression in particular at the level of the quantity of theproduct expressed.

[0019] A DNA sequence according to the invention is such that the codingnucleotide sequence that it controls is placed immediately downstream,in phase with the same reading frame as it or, on the other hand, it isseparated from this DNA sequence by a nucleotide fragment.

[0020] Hence the invention relates to a DNA sequence for the control ofthe expression of a coding sequence for a gene in a cell host, the DNAsequence is characterized in that it includes a promoter and anucleotide sequence or downstream region situated in particulardownstream of the promoter and upstream at said coding sequence. Thenucleotide sequence or downstream region contains a region essentiallycomplementary to the 3′ end of a bacterial ribosomal RNA. The DNAsequence of the invention is capable of intervening to enhance theexpression of the coding sequence placed downstream in a cell host.

[0021] The inventors have identified a DNA sequence of the typepreviously described, capable of intervening in the control of theexpression of the coding sequence of the cryIIIA gene, and making itpossible in particular to obtain a high level of expression when thecoding sequence is placed on a low copy number plasmid.

[0022] The invention also relates to a DNA sequence characterized by thefollowing properties:

[0023] it is included in a DNA sequence about 1692 bp long, defined bythe restriction sites HindIII-PstI (H₂-P₁ fragment), such as thatobtained by partial digestion of the 6 kb BamHI fragment borne by thecryIIIA gene of Bacillus thuringiensis strain LM79;

[0024] it is capable of intervening in the control of the expression ofa coding nucleotide sequence placed downstream in a host cell, inparticular a bacterial cell host of the Bacillus thuringiensis and/orBacillus subtilis type.

[0025] The restriction sites referred to above are shown in FIG. 1.

[0026] In the remainder of the text the abbreviations H_(n) will be usedto designate the HindIII site having the position “n” with respect tothe first HindIII site of the BamHI fragment. Similarly, the expressionP_(n) designates the PstI site at position “n” with respect to the firstPstI site on the BamHI fragment.

[0027] The DNA sequence defined above can be isolated and purified forexample from the plasmid bearing the cryIIIA gene of Bacillusthuringiensis.

[0028] The expression system for cryIIIA comprises a first nucleotidesequence or promoter situated between the TaqI and PacI sites (positions907 to 990) and a second nucleotide sequence or “downstream region”included between the XmnI and TaqI sites (positions 1179 to 1559) asshown in FIG. 6. The presence of two sequences of this type is preferredto obtain an optimal level of expression of the cryIIIA gene or ofanother gene placed under the control of this expression system.

[0029] Also included in the framework of the invention is an expressionvector characterized in that it is modified at one of its sites by a DNAsequence such as that described above so that said DNA sequenceintervenes in the control of the expression of a specific codingnucleotide sequence.

[0030] A vector of the invention may preferably be a plasmid, forexample a plasmid of the replicative type.

[0031] A particularly useful vector is the plasmid pHT7902′lacZdeposited with the CNCM (Collection Nationale de Cultures deMicro-organismes-Paris-France) on Apr. 20, 1993 under No. I-1301.

[0032] The object of the invention is also a recombinant cell hostcharacterized in that it is modified by a DNA sequence such as thatpreviously defined or by an expression vector described above. Aparticularly useful cell host is the strain 407-OA:Km^(R) (pHT305P)deposited with the CNCM on May 3, 1994 under No I-1412.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The object of the invention is a DNA sequence capable ofinfluencing the expression of the coding part of a gene in a bacterialcell host. More particularly, the invention relates to the associationof two nucleotide sequences, namely a promoter and a downstream regioncapable of intervening at the post-transcriptional level when the codingpart of the gene is expressed.

[0034] The expression system of the invention which, as will bedescribed in detail hereafter, probably involves the hybridization of apart of the downstream region with the 3′ end of the 16S RNA of abacterial ribosome, may be used for the expression of genes in a widerange of host cells. This extensive use of the expression system of theinvention is possible, given the considerable homology observed at thelevel of the various 16S RNAs of bacterial ribosomes. Since theinventors have defined the regions essential for its functioning, theexpression system of the present invention can thus be used in any typeof bacterial host, the necessary adaptations forming part of theknowledge of the specialist.

[0035] In general and without wishing to restrict it for reasons whichwill become evident below, the expression system of the presentinvention when used for the expression of genes in Gram⁺ bacteria of theBacillus type is situated upstream from the coding part of the gene tobe expressed. More particularly, the downstream region is normallysituated immediately upstream from the gene whereas the promoter islocated upstream from the downstream region, although another positionmight be envisaged for this latter. It is possible to envisage thedisplacement of the downstream region when the system is used in a cellhost of the E. coli type in which the mRNAs are degraded in the reversesense. It is possible to envisage the use of a downstream regiondownstream and upstream of the coding sequence which would permit the“protection” of the coding region by a mechanism which will be describedin detail below.

[0036] According to a first preferred embodiment of the invention, theDNA sequence corresponds to the HindIII-PstI (H₂-P₁) sequence describedabove and comprises two nucleotide sequences (a promoter and adownstream region) having distinct functions.

[0037] According to a particularly useful embodiment of the invention,the DNA sequence corresponds to the nucleotide sequence designated bythe expression Seq. No. b 1 and corresponding to the DNA fragmentcomprising the nucleotides 1 to 1692 of the sequence shown in FIG. 3.

[0038] The promoter and the downstream region of the DNA sequence of theinvention are described in detail below.

Nucleotide Sequences Exhibiting a Promoter Activity

[0039] Preferably, a DNA sequence of the invention intervenes at thelevel of the control of transcription.

[0040] In this case it is a nucleotide sequence previously identified asbeing the promoter. Generally speaking as mentioned previously, thepromoter is situated upstream from the downstream region and hence at acertain distance from the coding region of the gene. However, it ispossible to envisage the relocation of the promoter provided it remainslocalized upstream from the downstream region.

[0041] As to the nature of the promoter, it seems preferable to use apromoter derived from the host cell used for the expression of the geneof interest. However, in certain situations the use of an exogenouspromoter may be indicated. For example, promoters such as the promotersof the degO, λPL, lacZ, cryI, cryIV or α-amylene genes may be used.

[0042] In the context of the present invention particularly preferredfragments comprising a promoter region are the following fragments,shown in FIG. 1:

[0043] the sequence defined by the TaqI-PacI restriction sites; for thesake of convenience, PacI is taken to designate the end of this fragmentwhich is in reality found at nucleotide 990 of the sequence shown inFIG. 3, whereas the PacI site ends at position 985,

[0044] or any fragment of this sequence, which conserves the propertiesof this sequence with respect to the control of the expression of codingnucleotide sequence.

[0045] More particularly, any part of at least 10 nucleotides of thissequence, naturally consecutive or not, capable of intervening in thecontrol of the expression of a coding nucleotide sequence placeddownstream in a cell host constitutes a preferred embodiment of theinvention. For example, within the sequence mentioned previously arefound the −35 (TTGCAA) and −10 (TAAGCT) boxes of the promoter.

[0046] According to another embodiment of the invention the “control”DNA sequences comprising the promoter mentioned above are characterizedby their nucleotide sequence. In this respect, the object of theinvention in particular is the DNA sequences corresponding to thefollowing sequences:

[0047] the DNA sequence corresponding to the Seq No. 3 sequencecorresponding to the fragment comprising the nucleotides 907 to 990 ofthe sequence shown in FIG. 3, or a variant comprising the nucleotides907 to 985.

[0048] The object of the invention is also DNA sequences hybridizingunder non-stringent conditions, such as those defined below, with one ofthe sequences described above. In this case, one of the above sequencesin question is used as probe.

Sequences of the Downstream Region

[0049] A sequence of the invention included in the downstream region isselected for its capacity to intervene in order to enhance theexpression of a gene which would be initiated by a promoter situatedupstream from this sequence. It is probably a sequence capable ofintervening at the post-transcriptional level when the coding sequenceis expressed.

[0050] In fact, the experimental results obtained by the inventors seemto indicate that the post-transcriptional effect of the downstreamregion previously defined results, at least when the cryIIIA gene isbeing expressed, from the hybridization between the 16S ribosomal RNA ofthe host cell and an S2 sequence of the cryIIIA messenger RNA. It seemsthat the ribosome or a part of the ribosome binds to this downstreamregion and thus protects the mRNA from exonuclease degradation initiatedat the 5′. This binding is thus expected to have the effect ofincreasing the stability of the messengers and of thus enhancing thelevel of expression of the cloned gene.

[0051] One of the particularly preferred fragments in the context of theembodiment of the invention and one which may be used as downstreamregion is the following fragment, shown in FIG. 1:

[0052] the sequence defined by the restriction sites XmnI-TaqI(positions 1179 to 1556),

[0053] or any fragment of this sequence conserving the properties ofthis sequence with respect to the control of the expression of a codingnucleotide sequence.

[0054] According to another embodiment of the invention, the “control”DNA sequences comprising the downstream region mentioned above arecharacterized by their nucleotide sequence. In this respect, the objectof the invention is in particular the DNA sequences corresponding to thefollowing sequences:

[0055] the DNA sequence corresponding to the sequence Seq No.4corresponding to the fragment comprising the nucleotides 1179 to 1559 ofthe sequence shown in FIG. 3,

[0056] the DNA sequence corresponding to the sequence Seq No.5corresponding to the fragment comprising the nucleotides 1179 to 1556 ofthe sequence shown in FIG. 3,

[0057] the DNA sequence corresponding to the sequence Seq No.11corresponding to the fragment comprising the nucleotides 1413 to 1556 ofthe sequence shown in FIG. 3,

[0058] the DNA sequence corresponding to sequence Seq No.8 correspondingto the fragment comprising the nucleotides 1413 to 1461 of the sequenceshown in FIG. 3,

[0059] the DNA sequence corresponding to the sequence Seq No.9corresponding to the following DNA fragment:5′-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3′3′-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′

[0060] the DNA sequence corresponding to the sequence Seq No.10corresponding to the following DNA fragment:5′-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3′3′-GAACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′

[0061] The object of the invention is also DNA sequences hybridizingunder non-stringent conditions such as those defined hereafter, with oneof the sequences described above. In this case, the relevant sequencedefined above is used as probe.

[0062] It seems that the downstream region consists initially of aregion said to be “essential”, sufficiently complementary to the 3′ endof a 16S bacterial ribosomal RNA to allow the binding of the ribosome tothis essential region. Downstream from this essential region bearing theribosomal binding site, a second region is assumed to be situatedcomprising an additional structure capable of having an additionalpositive effect at the level of the expression of the coding sequence.It is possible that this second sequence prevents the movement of theribosome once this latter is bound to the essential region.

[0063] For example, in the expression system of the cryIIIA gene, itseems that the nucleotide sequence situated between the positions 1413and 1556 of the sequence shown in FIG. 3 comprises the region essentialfor ribosomal binding as well as the second region downstream from thebinding site. Although the second region is not absolutely essential forobtaining an enhanced expression of the coding sequence, it seems thatits deletion reduces the expression yields. In fact, experimentalresults have shown that the deletion of the region situated between thenucleotides 1462 and 1556 of the sequence shown in FIG. 3 leads to aslight diminution of the expression of the coding sequence.

[0064] It seems that the minimal length of the nucleotide sequencemaking possible adequate binding to the ribosome is about 10nucleotides. The object of the invention is thus also any part of atleast 10 nucleotides of the H₂-P₁ sequence, naturally or notconsecutive, capable of controlling in a cell host of the Bacillus typethe expression of a coding nucleotide sequence placed downstream or thispart of the H₂-P₁ sequence.

[0065] In the specific case of the expression system of the cryIIIAgene, it would seem that the sequence of the “essential” regionincluding the binding site is the following: 5′ - GAAAGGAGG - 3′ 3′ -CTTTCCTCC - 5′

[0066] It is possible to make minor modifications at the binding site inas much as the intensity of the interaction between the 3′ end of the16S ribosomal RNA and this “essential” region is sufficiently strong forthere to be hybridization between the ribosome and the binding site.From the calculations of the interaction energy which may be carried outby the specialist skilled in the art, modifications to the binding sitecan be envisaged if the intensity of the binding remains about the sameas the the intensity measured when the natural “essential” region isused.

[0067] In the case of the binding site previously illustrated, it ispossible to envisage certain modifications to the first four nucleotidesas well as to the seventh nucleotide. However, it seems that thenucleotides in positions 5, 6, 8 and 9 are important for maintaining anappropriate intensity of interaction during hybridization with the 16Sribosomal RNA.

[0068] Since the 3′ end of the 16S bacterial ribosomal RNA is relativelywell conserved from one bacterial species to another, the expressionsystem of the present invention may thus be used in a large number ofbacterial hosts without substantial modifications having to be made.

[0069] The object of the invention is thus also a DNA sequencecharacterized by the following properties:

[0070] it is contained in a nucleotide sequence hybridizing undernon-stringent conditions with the DNA fragment included between thenucleotides 1413 and 1559 of the sequence shown in FIG. 3;

[0071] it is capable of intervening in the control of the expression ina host cell of a coding sequence, in particular a sequence coding for aBacillus polypeptide, toxic towards insects or a sequence coding for apolypeptide expressed during the stationary phase in Bacillus.

[0072] A sequence coding for a Bacillus polypeptide, toxic towardsinsect larvae is for example a sequence included in the cryIIIB gene ofB. thuringiensis.

[0073] A DNA sequence corresponding to this definition can be identifiedby using oligonucleotide primers.

[0074] Hybridization under non-stringent conditions between the test DNAsequence and the DNA fragment included between the nucleotides 1413 and1559 of the sequence of FIG. 3 used as probe will be conducted asfollows:

[0075] The DNA probe and the sequences bound to the nitrocellulosefilter or to the nylon filter are hybridized at 42° C. for 18 h withshaking in the presence of formamide (30%), 5× SSC of the 1× Denhardtsolution. The 1× Denhardt solution is composed of 0.02% Ficoll, 0.02%polyvinylpyrrolidone and 0.02% bovine serum albumin. The 1× SSC iscomposed of 0.15M NaCl and 0.015 M sodium citrate. After hybridization,the filter is successively washed at 42° C. for 10 minutes in each ofthe following solutions:

[0076] formamide (30%), 5× SSC

[0077] 2× SSC

[0078] 1× SSC

[0079] 0.5× SSC

[0080] The hybridization conditions just described are those which areused for all the applications of the present invention when necessary.

[0081] The DNA sequences according to the invention may be optionallyrecombinant among themselves or associated on a vector at differentsites. In particular, the TaqI-PacI fragment is advantageouslyassociated with the XmnI-TaqI fragment defined above in the form of asingle sequence and also the TaqI-PacI fragment with the sequence SeqNo.8. Such sequences have the advantageous property of making possible ahigh level of expression (up to 60,000 Miller units) of the codingnucleotide sequence, a level of expression which may be observed withthe beta-galactosidase gene.

[0082] Furthermore, particularly preferred fragments in the context ofthe embodiment of the invention are the following fragments shown inFIG. 8B:

[0083] the sequence defined by the TaqI-TaqI restriction sites,

[0084] or any fragment of these sequences conserving the properties ofthese sequences with respect to the control of the expression of anucleotide coding sequence.

[0085] According to another embodiment of the invention, the DNAsequences referred to above are characterized by their nucleotidesequence. In this respect, the object of the invention is in particularthe DNA sequences corresponding to the following sequences:

[0086] the sequence Seq No.2, corresponding to the fragment comprisingthe nucleotides 907 to 1559 of the sequence shown in FIG. 3,

[0087] the DNA sequence corresponding to the sequence Seq No.6corresponding to the fragment comprising the nucleotides 907 to 1353 and1413 to 1556 of the sequence shown in FIG. 3,

[0088] the DNA sequence corresponding to the sequence Seq No.7corresponding to the fragment comprising the nucleotides 907 to 990 and1179 to 1559 of the sequence shown in FIG. 3.

[0089] The object of the invention is also DNA sequences hybridizingunder non-stringent conditions such as those defined above with one ofthe sequences described above. In this case, one of the above sequencesis used as probe.

[0090] The DNA sequences of the invention can be isolated and purifiedfrom Bacillus, in particular from B. thuringiensis; they can also beprepared by synthesis according to known procedures.

[0091] Also included in the framework of the invention are the RNAsequences corresponding to the DNA sequences described above.

[0092] The object of the invention is also a recombinant DNA sequencecharacterized in that it comprises a defined coding sequence under thecontrol of a DNA sequence corresponding to one of the precedingspecifications.

[0093] The capacity of the DNAs of the invention to intervene in thecontrol of the expression of nucleotide sequences can be verified byimplementing the following test:

[0094] the DNA sequence of the invention whose capacity to intervene inthe control of the expression of a coding sequence it is desired toevaluate is inserted in a low copy number plasmid upstream from a codingnucleotide sequence.

[0095] the plasmid thus prepared is used to transform (for example byelectroporation) a strain of Bacillus thuringiensis for example a B.thuringiensis strain HD1 cry⁻B;

[0096] the Bacillus strain thus transformed is cultured under conditionspermitting the expression of the coding nucleotide sequence;

[0097] the expression product of this coding nucleotide sequence isdetected by current qualitative and/or quantitative measuringprocedures.

[0098] In order to carry out this test, the coding nucleotide sequenceshould advantageously be the coding sequence of the cryIIIA gene ofBacillus thuringiensis or for example a sequence coding forbeta-galactosidase.

Cell Hosts

[0099] Different types of cell host may be used in the framework of theinvention. Mention should be made as an example of Bacillus, for exampleBacillus thuringiensis or Bacillus subtilis. It is also possible toenvisage the use of cells such as E. coli.

[0100] In cell hosts capable of sporulating, the coding sequence may beexpressed during the vegetative phase or the stationary phase of growthor during sporulation.

[0101] A interesting cell host in the framework of the invention mayalso be constituted by a vegetal or animal cell.

[0102] If it is necessary or desired, depending on the nature of thecoding nucleotide sequence expressed, a signal sequence can also beinserted in the expression vector of the invention so that theexpression product of the coding sequence is exposed at the surface ofthe cell host, or even exported from this cell host.

[0103] In a really interesting manner it will be possible to use strainsof Bacillus which have become asporogenic either naturally or as aresult of mutation and in particular strains of Bacillus subtilis orBacillus thuringiensis.

[0104] Since the inventors have demonstrated that the DNA sequences ofthe invention permit the expression of a defined coding sequenceindependently of the sporulation phase of strains of the Bacillus type,an asporogenic host may offer the advantage of providing agents ofexpression of coding sequences to be included in biopesticidecompositions whose possible negative effects vis-a-vis the environmentwould be expected to be attenuated, and even eliminated.

[0105] The asporogenic host selected is particularly advantageous forexpressing a coding sequence during its stationary phase of growth, whenthe coding sequence is under the control of one of the sequences of theinvention.

[0106] In the case of asporogenic strains of Bacillus obtained bymutation, an example illustrating the particular efficacy of this typeof strain for the expression of a coding sequence during the stationaryphase of growth is the construction of a B. thuringiensis strain mutatedin the SpoOA gene. A B. thuringiensis strain in which the spoOA gene isinactivated and which bears a gene, for example a gene for aninsecticidal toxin cryI, cryII, cryIII or cryIV or also a gene ofindustrial interest whose expression is placed under the control of thecryIIIA expression system offers advantageous characteristics. Inparticular, the B. thuringiensis strain 407.OA:Km^(R) ((pHT305P) whoseconstruction is described in detail below has at least the followingadvantages:

[0107] a) oveproduction of proteins during the stationary phase ofgrowth;

[0108] b) the proteins (for example, biopesticides) remain enclosed inthe cell and thus would be expected to have an increased persistence inthe environment; and

[0109] c) the potential problems linked to the dissemination of sporesare thus avoided.

[0110] Other characteristics and advantages of the invention follow fromthe Examples which follow as well as from the Figures:

[0111]FIG. 1: Schematic restriction map of the plasmids used(A)—Physical map of the shuttle vector pHT304. The arrows above Erm^(R)and Ap^(R) indicate the direction of transcription of the ermC and blagene, respectively. The arrow and the expression LacZ indicate thedirection of transcription from the promoter of the LacZ gene. ori Bt isthe replication region of the plasmid pHT1030 of B. thuringiensis(B)—Simplified restriction map of the fragments bearing the cryIIIAgene. The A fragment is a 6 kb BamHI fragment of B. thuringiensis LM79;the restriction fragments G, P and H were obtained by partial digestionwith HindIII and C was obtained after total digestion of fragment A withHindIII. These fragments were cloned in pHT304 to give the derivativespHT305A, pHT305G, pHT305P, pHT305H and pHT305C, respectively. ThecryIIIA gene (hatched box) and the direction of transcription areindicated. The numbers under each site indicate their order from left toright.

[0112]FIG. 2: Analysis of the proteins of the transformants of B.thuringiensis expressing the cryIIIA gene. An identical volume (20 μl)of samples was loaded into each well. The lines 1 to 4 and 6 to 8 of B.thuringiensis Kurstaki HD1 Cry⁻ B bearing pHT305A, pHT305G, pHT305H,pHT305P, pHT305HΩH₂-H₃, pHT305C and pHT304, respectively. Column 5corresponds to the molecular weight markers (from top to bottom 97, 66,60, 43 and 30 kDa). The arrows indicate the crystal components of 73 and67 kDa.

[0113]FIG. 3: Nucleotide sequence of the 5′ end of the region upstreamfrom the cryIIIA gene.

[0114] (A)—Physical map of the H₂-P₁ (H₂-H₃+H₃-P₁) fragment in the 5′ to3′ orientation. The positions of the nucleotides of the two HindIIIsites (H₂+H₃) which define the grey tinted fragment are indicated. Thesecond sequenced segment (H₃-P₁ fragment) was the fragment between thethird HindIII site and the PstI site (P1). An ATG transcriptioninitiation site for the CryIIIA toxin is shown. The numbering of thenucleotides is reported with respect to the sequenced fragment and notwith respect to the initiation of transcription.

[0115] (B)—Nucleotide sequence of the fragment H₂-P₁. The ATG initiationcodon is indicated in bold characters and the end of the majortranscript on the gel, specific for the cryIIIA, corresponds to the Tlocated at position 1413 Another transcript starts at nucleotide 983.;it is apparently a minor component on the gel. The sequence comprises atleast two inverted repeats. The numbering of the nucleotides starts fromthe second HindIII site and ends at the PstI site shown in FIG. 3A.

[0116]FIG. 4: Representation of the plasmids PAF1, pHT304′lacZ,pHT7901′lacZ and pHT7902′lacZ.

[0117]FIG. 5: Profile of beta-galactosidase activity. The growth of theBt cells and the conditions for preparing the samples as well as thetest are described in “Materials and Methods”. the time t₀ indicates theend of the exponential phase and t_(n) is the number of hours before (−)or after time zero.

[0118]FIG. 6: Detailed restriction map of the plasmids pHT7902′lacZ,7903′lacZ, 7907′lacZ, 7909′lacZ, 7930′lacZ and 7931′lacZ. These plasmidswere inserted into B. thuringiensis and the beta-galactosidase activitywas measured at time t₆ of sporulation (in Miller units). The activitiesof 30,000, 30,000, 3.500, 2,000, 35,000 and 60,000 respectively areobserved.

[0119]FIG. 7: Beta-galactosidase activity in B. subtilis strains Spo⁻and Spo⁺; the cultures are grown in SP medium.

[0120]FIG. 8: Schematic restriction map of the constructions used tomeasured the transcriptional activity of the regions of the expressionsystem at cryIIIA in B. thuringiensis strain kurstaki HD1 Cry⁻B.

[0121] A—Physical map of the vector pHT304-18Z. The arrows indicate thedirection of transcription of the genes ermC, bla, lacZ and the promoterplacZ; and the orientation of the replication in E. coli (oriEc).ori1030 indicates the region of replication of the plasmid pHT1030(Lereclus and Arantes, Mol. Microbiol. 1992, 7: 35-46). SD indicates theribosomal binding site of the spoVG gene placed in front of the lacZgene (Perkins and Youngman, 1986, Proc. Natl. Acad. Sci. USA, 83:140-144).

[0122] B—Physical representation and transcriptional activity of thedifferent regions of the cryIIIA expression system fused with the lacZgene. The numbering of the nucleotides is established according to theDNA sequence of the H₂-P₁ fragment presented in FIG. 3B. The arrowsindicate the position of the 5′ ends of the transcripts as they areidentified by primer extension. The dotted lines indicate thelocalization of the deleted fragments. The beta-galactosidase activityof the different constructions was measured at times t₀ and t₆ ofsporulation and is indicated in Miller units.

[0123]FIG. 9: Determination of the 5′ end of the cryIIIA/lacZ transcriptproduced by the B. thuringiensis strain bearing the plasmidpHT7815/8′lacZ. The total RNA of the cells was extracted at t₃ andsubjected to a primer extension experiment with the reversetranscriptase using as primer the following oligonucleotide:5′-CGTAATCTTACGTCAGTAACTTCCACAG> −3′. This oligonucleotide iscomplementary to the region localized between the ribosomal binding siteof the spoVG gene and the initiation codon of the lacZ gene. The sameoligonucleotide was used to determined the nucleotide sequence of thecorresponding region of the plasmid pHT7815/8. The 5′ end is numberedaccording to the DNA sequence of the H₂/P₁ fragment presented in FIG.3B.

[0124]FIG. 10: Schematic physical map of the constructions used tomeasure the post-transcriptional activity of the downstream region ofthe cryIIIA expression system in B. subtilis strain 168. The numberingof the nucleotides is established according to the DNA sequence of theH₂-P₁ fragment presented in FIG. 3B. The arrow indicates the startingposition of transcription located at position +984. The asterisk atposition 1421 indicates the replacement of GGA by CCC. The dashed linesindicate the location of the deleted DNA fragments. Thebeta-galactosidase activity of the different constructions was measuredat the time t₃ of sporulation and is indicated in Miller units.

[0125]FIG. 11: Nucleotide sequence of the spoOA gene of B. thuringiensisstrain 407.

[0126] A—Schematic restriction map of the 2.4 kb DNA fragment bearingthe spoOA gene. The arrow indicates the orientation of the transcriptionof the spoOA gene.

[0127] B—Nucleotide sequence of the open reading frame comprising thecoding sequence of the spoOA gene. The initiation codon GTG is indicatedin bold characters. The two HincII sites are underlined. The three dotsrepresent the stop codon.

MATERIALS AND METHODS Bacterial Strains and Growth Conditions

[0128]Escherichia coli K-12 TG1 [Δ(lac-proAB) supE thi hdsD (F′ traD36proA⁺ proB⁺ lacI

lacZΔDM15)] Gibson, T. J. et al. 1984 Thesis, University of Cambridge,Cambridge was used as host for the construction of the plasmidsrepresented in FIG. 1B and for the bacteriophage M13.

[0129]E. coli MC1061 {hsdR mcrB araD139Δ (araABC-leu) 7679 Δ lacX74 galUgalK rpsL thi} (Meissner, P. S. et al., 1987 Proc. Natl. Acad. Sci. USA84: 4171-4175) was used as host for the construction of the plasmidsshown in FIG. 7.

[0130]B. thuringiensis strain LM 79 which contains the cryIIIA gene wasisolated and characterized by Chaufaux J. et al. 1991. INRA colloquia58: 317-324.

[0131] This strain belongs to the serotype 8 and produces quantities oftoxins similar to those produced by other strains of B. thuringiensisbearing the cryIIIA gene (Donovan, V. P. et. al. 1988 Mol. Gen. Genet.214, 365-372-Sekar, V. et al. 1987 Proc. Natl. Acad. Sci. USA 84:7036-7040).

[0132]B. thuringiensis of the subspecies Kurstaki HD1 Cry⁻B was used ashost for the studies of regulation of the cryIIIA gene. The E. colistrains were cultured at 37° C. in a Luria medium and transformedaccording to the method described by Lederberg and Cohen (1974Bacteriol. 119: 1072-1074).

[0133] The B. thuringiensis strain subspecies Kurstaki HD1 Cry⁻B wascultured and transformed by electroporation according to the proceduredescribed by Lereclus et al. (1989 FEMS Microbiol. Lett. 60: 211-218).

[0134] The antibiotic concentrations for the selection of the bacteriawere 100 μg/ml for ampicillin and 25 μg/ml for erythromycin.

Construction of the Plasmids

[0135] The 6 kb BamHI fragment bearing the cryIIIA gene and the adjacentregions was isolated from B. thuringiensis LM79 and inserted into theunique BamHI site of pUC19 to produce pHT791 which was employed as DNAsource for the construction of the various plasmids used here. Theplasmid pHT305A was obtained by insertion of the 6 kb BamHI fragmentinto the unique BamHI site of the shuttle vector pHT304 (Arantes, O andLereclus D 1991, Gene 108: 115-119) (FIG. 1A). Samples of the 6 kb BamHIfragment were partially or completely digested with HindIII and theresulting fragments were cloned between the BamHI and HindIII sites orat the HindIII site of pHT304 to give the derivatives pHT305G, pHT305H,pHT305P and pHT305C (FIG. 1). The plasmid pHT305HΩH₂H₃ was obtained byinserting the H₂-H₃ fragment filled at the ends in the SmaI site ofpHT305H (fragment defined respectively by the second and third HindIIIsites of the 6 kb fragment).

[0136] The 45 kb SmaI-KpnI fragment of the pTV32 plasmid (Perkins, J. B.et al; 1986 Proc. Natl. Acad. Sci. USA 83: 140-144) containing the lacZand ermC genes was cloned in pEB111 (Leonhardt, H. et al. 1988 J. Gen.Microbiol. 134: 605-609) to give the plasmid pMC11. The plasmidpHT304′lacZ used to construct the transcriptional fusions was obtainedby cloning the 3.2 kb DraI-SmaI restriction fragment containing the lacZgene lacking a promoter isolated from pMC11, at the unique SmaI site ofpHT304. The plasmid pHT7901′lacZ was obtained by cloning the H₃-P₁fragment {(HindIIl-PstI) see FIG. 3A} between the unique HindIII andPstI sites of pHT304′lacZ. The plasmid pHT7902′lacZ was constructed bycloning the H₂-H₃ fragment (FIG. 3A) into the unique HindIII site ofphT7901′lacZ. The orientation of the H₂-H₃ fragment was determined bymapping the HpaI and BalI restriction sites with respect to the PstIsite. Two HpaI sites are located at the nucleotide positions of 50 and392; the BalI site is located at nucleotide position 670 (FIG. 3). Thegeneral structure of the recombinant plasmids bearing the lacZ fusion isgiven in FIG. 4.

DNA Manipulations

[0137] The standard procedures were used to extract the plasmids from E.coli to transfect the recombinant DNA of phage M13 and to purify thesingle-stranded DNA (Sambrook J et al., 1989 A laboratory manual, 2nded. Cold Spring Harbor Laboratory-Cold Spring Harbor, N.Y.). Therestriction enzymes, the T4 DNA ligase and the T4 polynucleotide kinasewere used in accordance with the manufacturer's instructions. The Klenowfragment of the DNA polymerase I and deoxyribonucleoside triphosphateswere used to provide the H₂-H₃ fragment with blunt ends. The DNArestriction fragments were purified on agarose gels using the Prep Agene kit (Bio-Rad). The nucleotide sequences were determined by thedideoxy chain termination method (Sanger F. et al. 1977 Proc. Natl.Acad. Sci. vol. 175, 1993 USA 74: 5463-5467) using the M13mp18 andM13mp19 phages as matrices as well as the Sequenase kit version 2.0 (USBiochemical Cor. Cleveland Ohio) and {α-³⁵S} dATP (15 TBq; Amersham,United Kingdom).

Computer Analysis

[0138] The DNA sequences were analysed by using the programs of thePasteur Institute on a general data-processing computer MV10000.

Extraction of the RNA Extension of the Primers, Northern Analysis of theRNA and Dot Blot Analysis

[0139] The B. thuringiensis subspecies Kurstaki HD1 Cry⁻B (pHT305P) wascultured in a HCT medium (Lecadet et al. 1980 J. Gen. Microbiol. 121:203-212) at 30° C. by shaking. The samples were taken at t₀, t₃, t₆ andt₉ (t₀ is defined as being the start of sporulation and t_(n) indicatesthe number of hours after the start of sporulation). The cells wererecovered by centrifugation, resuspended in a HCO medium (Lecadet, M. M.et al., 1980 J. Gen Microbiol. 121: 203-212) containing 50 mM of sodiumazide and immediately frozen at −70° C. until the RNA was extracted(Glatron, M. F. et al., 1972, Biochemie 54: 1291-1301). For theelongation test of the primer, a first oligonucleotide—a 39-mer (3′-CTTAGG CTT GTT AGC TTC ACT TGT ACT ATG TTA TTT TTG-5′) complementary to theregion 3′-1544 to 1583-5′ of the cryIIIA gene was synthesized and its5′0 end was labeled with (γ-32P) dATP (110 TBq/mmol) by the T4polynucleotide kinase. The 39-mer oligonucleotide was purified on acolumn of Sephadex G-25 (Pharmacia) (incorporation about 70%) and to beused as primer it was mixed with 50 μg of total RNA.

[0140] A second oligonucleotide, a 32-mer complementary to the regionlocated between the positions 1090 and 1121 was also used as primer andmade possible the detection of a second transcript, the start oftranscription of which is situated at position 983. This oligonucleotidecorresponds to the sequence

[0141] 5′-GTTAGATAAGCATTTGAGGTAGAGTCCGTCCG-3′

[0142] The hybridization (at 30° C.), the extension of the primer andthe analysis of the products were carried out as described byDebarbouillé, M et al., (1983, J. Bacteriol. 153: 1221-1227). Theprimers of the 39-mer and the 32-mer were used for the elongation of thefragment H₃-P₁ cloned in M13mp19 and for the elongation of the H₂-P₁fragment cloned in pHT7902′lacZ, respectively. The products resultingfrom the reactions were placed on gels in parallel with transcriptionproducts to determine the 5′ ends of the transcripts.

[0143] A Northern blot analysis was performed with denatured RNAfractionated by electrophoresis on agarose gels containing 1.5%formaldehyde and transferred in a vacuum to Hybond-N⁺ (Amersham)membranes in 20×SSC for 1 h (1×SSC corresponds to 150 mM NaCl plus 15 mMsodium citrate, pH 7.0). The PstI-EcoRI restriction fragment of 874 bp(internal to the cryIIIA gene) was labelled with ³²P with a nicktranslation kit (Boehringer Mannheim), then denatured and used as probe.A prehybridization was performed at 42° C. for 4 hours in a mediumcontaining 50% formamide-1M NaCl-1% sodium dodecyl sulfate(SDS)-10×Denhardt's solution-50 mM Tris HCl (pH 7.5) −0.1% sodium PP,denatured salmon sperm DNA (>100 μg/ml) and the labelled probe (10⁸cpm/μg) was added to the prehybridization solution and the incubationwas continued overnight. The membrane was washed at 65° C. for 30minutes twice with 2×SSC-0.5% SDS, and once with 0.5×SSC-0.5% SDS.

[0144] Equal quantities of RNA of synchronous cultures of B.thuringiensis subspecies Kurstaki HD1 Cry⁻B bearing the plasmids pHT305Por pHT305H taken at t₃ were deposited on to Hybond-C Extra membranes(Amersham) with a manifold apparatus (Schleicher & Schueller) by usingthe dot blot protocol described by Sambrook et al. (Sambrook, J. et al.1989 Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The probeand the hybridization conditions were those described in the Northernblot tests.

Preparation of the Crystal and Analysis

[0145] The cells were cultured in a HCT medium at 30° C. with shakingfor 48 hours and the crystals were prepared according to the methoddescribed in the publication by Lecadet, M. M. et al. (1992 Appl.Environ. Microbiol. 58: 840-849) with the exception of the fact that theNaCl concentration was 150 mM. For gel electrophoresis onpolyacrylamide-SDS (PAGE) 20 μl of each sample were used (Lereclus, D.et al. 1989 (FEMS Microbiol. Lett. 66: 211-218).

Test for the Detection of Beta-galactosidase

[0146] The strains of E. coli and B. thuringiensis containing the lacZtranscription fusions were detected by depositing on the solid mediumthe chromogenic substrate5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (40 μg/ml) andsuitable antibiotics. The isolated strains were cultured as indicatedand recovered at t-₂, t-₁, t₀, t_(1.5), t₃, t_(4.5), t₆ and t_(7.5).After centrifugation, the pellets were immediately frozen at −70° C. (inorder to prevent the inactivation of the beta-galactosidase) and thawedjust before the treatment with ultrasonics to detect thebeta-galactosidase (Msadek, T. et al. 1990 J. Bacteriol. 172: 824-834).The specific activities presented (expressed in Miller units permilligram of protein) correspond to the mean values of at least twoindependent experiments.

RESULTS The Expression of the CryIIIA Gene Requires the Presence of aDNA Fragment Upstream From the Gene

[0147] Arantes and Lereclus (1991 Gene 108: 115-119) have shown that thecryIIIA gene was only weakly expressed in the B. thuringiensis strainHD1 Cry⁻B when it was cloned in a low copy number vector such as pHT304(4 copies per chromosome equivalent).

[0148] Starting from a 6 kb BamHI fragment bearing the cryIIIA gene andthe adjacent regions (FIG. 1B) isolated from the B. thuringiensis strainLM79 specific for the Coleoptera, it has been investigated whetherregions upstream from the gene might be implicated in the regulation ofthe expression of this gene. The 6 kb fragment was cloned into theunique BamHI site of the vector pHT304 (FIG. 1A); fragments obtainedafter partial or total digestion by HindIII of the 6 kb BamHI fragmentwere also inserted independently in the same plasmid to give thederivatives pHT305A, pHT305G, pHT305H, pHT305P and pHT305C (FIG. 1B).The five recombinant plasmids were then introduced in B. thuringiensissubspecies Kurstaki HD1 Cry⁻B by electroporation and the transformantswere cultured for two days at 30° C. in a HCT medium (Lecadet, M. M. etal. 1980 J. Gen. Microbiol. 121: 203-212) containing 25 μg oferythromycin per ml.

[0149] Preparations of spores containing crystals were recovered fromcultures and examined by phase contrast microscopy and SDS-PAGE (FIG.2). The recombinant strains bearing the vectors pHT305A, pHT305G andpHT305P (FIG. 2, lines 1, 2 and 4 respectively) produced largequantities of a flat rhomboid crystal characteristic of strains activeagainst the larvae of the Coleoptera,. The principal components of thesecrystals were two peptides of about 73 and 67 kDa such as thosepreviously described for the B. thuringiensis strains bearing cryIIIA(Donovan, W. P. et al. 1988 Mol. Gen. Genet. 214: 365-372).

[0150] On the other hand, no production of crystal was detected with thestrains bearing pHT305H or pHT305C (FIG. 2, lines 3 and 7 respectively).The hypothesis has been put forward that these plasmids lack certainelements present, conversely, in the derivatives pHT305A, pHT305G andpHT305P. This possible additional element is situated ona 1 kb DNAfragment between the second and third HindIII site, this fragment beingdesignated by H₂-H₃ (FIG. 1B). In order to test whether its activatingeffect depended on its position, the H₂-H₃ fragment was ligated to aSmaI site of pHT305H. In the resulting plasmid (pHT305HΩH₂H₃), the H₂H₃plasmid is located downstream from the cryIIIA gene. The synthesis ofthe CryIIIA toxin pHT305HΠH₂H₃ proved to be as weak as with the plasmidpHT305H (FIG. 2, line 6). This absence of effect might be due to eitherthe new location of the H₂-H₃ fragment, this location beinginappropriate or to the disorganization of its functional structure. Inthis case, the functional element starting within the H₂-H₃ fragmentwould be extended to a region beyond the HindIII site described andwould potentially comprise the region of the promoter.

Sequencing of the DNA and Analysis

[0151] The nucleotide sequence of the 979 b H₂-H₃ fragment of theplasmid pHT791 was determined (FIG. 3B). Furthermore, the sequence of713 bp extending from the third HindIII site to the first PstI site(H₃-P₁ fragment) was determined (FIG. 3B). This second fragment bearsthe region upstream of the promoter, the promoter itself, the potentialribosomal binding site and the first 151 codons of the cryIIIA gene(Sekar, V et al., 1987 Proc. Natl; Acad. Sci. USA 84: 7036-7040). Thereis no difference between the sequence of the H₃-P₁ fragment isolatedfrom the strain LM79 and the corresponding regions of the cryIIIA genesisolated from B. thuringiensis subspecies tenebrionis, B. thuringiensissubspecies san diego and the strain EG2158 (Donovan W. P. et al.,Herrnstadt C. et al., Höfte H. J. et al., Sekar V. et al.). No sequencepotentially coding for a protein other than that corresponding to the 5′end of cryIIIA was found. This region exhibits a high proportion of A+Tbases (adenine-plus-thymine) corresponding to about 81% between thebases 770 and 990 and two inverted repeat sequences. The first invertedrepeat sequence is imperfect (16 of the 17 bp are identical) with acentre of symmetry at nucleotide 858 and the second is a perfectinverted repeat of 12 bp with a centre of symmetry at nucleotide 1379.The free energies leading to the formation of the stem loop structurescalculated according to the method of Tinoco et al. (Tinoco, J. J. etal., 1973 Nature (London) New Biol. 246: 40-41) were −57.7 and −66.1kJ/mol., respectively.

Analysis of the Initiation Site and the Duration of Transcription in thePresence of the H₂-H₃ Fragment

[0152] Sekar et al. have mapped the initiation site of the transcriptionof the cryIIIA gene starting from the RNAs isolated from early phasecells (stage II) and intermediary phase cells (stages III to IV) ofsporulation by using the mung beam nuclease. These periods of growthcorrespond to t₂ to t₅. The extension from the primers was performed onRNAs extracted from cells in culture at t₀, t₃, t₆ and t₉ to determinewhether other initiation sites are involved during the early and latephases of growth and in order to determine at which stage maximaltranscription occurs. A start site for transcription appeared in theform of a weakly radioactive signal in the samples taken at t₀ and t₉and this signal proved to be more intense in the samples at t₃ and t₆.This initiation site of transcription was mapped one nucleotide upstreamfrom that described by Sekar et al.

[0153] These results show that the major transcript has as its 5′ endthe T located at position 1413 (FIG. 3). However, the T located atposition 1413 (FIG. 3) might constitute the end of a stable messengerwhose true initiation site is located upstream.

Detection and Quantitative Analysis of the Specific mRNA of the CryIIIAToxin in the Presence of the H₂-H₃ Fragment

[0154] The reverse transcriptase is satisfactory for extension from theprimers in the case of fragments containing only 100 to 150 bases in asmuch as this enzyme may stop or be interrupted in regions containingconsiderable secondary structures at the level of the RNA matrix. Inorder to study the presence of a potential initiation site fortranscription located very far upstream from the 5′ end of the cryIIIAgene, a Northern blot analysis was performed. The total RNA of thestrain bearing pHT305P was recovered at t₀, t₃, t₆ and t₉. The RNAs wereseparated by electrophoresis on agarose gels and hybridized with a probecorresponding to the labelled internal fragments of cryIIIA (PstI-EcoRIfragment at 874 bp). In all of the samples a principal transcript ofabout 2.5 kb was detected. This is consistent with the size of thetranscript defined by the initiation site for transcription describedabove and a potential termination sequence located about 400 bpdownstream from the stop codon of cryIIIA described by Donovan et al.

[0155] The relative quantities of specific mRNA of the CryIIIA toxinsynthesized by the strain bearing pHT305P and by the strain bearingpHT305H were compared by a dot blot procedure RNAs isolated fromsynchronous cultures recovered at t₃ were immobilized on anitrocellulose membrane and hybridized with an excess of PstI-EcoRIprobe of cryIIIA. The strain bearing pHT305P contained about 10 to 15times more mRNA specific for cryIIIA than the strain containing pHT305H.

Production of Beta-galactosidase from the Fusion of H₂-H₃:: lacZ

[0156] The relative synthesis of the cryIIIA transcript in the presenceand in the absence of the H₂-H₃ fragment indicated that this DNA segmentregulates the expression of the cryIIIA gene at the level of thetranscription rather than at the level of translation. Fusion with thelacZ gene was carried out to test the effect produced on transcriptionby the H₂-H₃ fragment. The lacZ gene lacking the promoter was subclonedin to the SmaI site of pHT304. The resulting plasmid pHT304′lacZconstitutes a system making it possible to generate fusion transcriptsand to study their expression in B. thuringiensis under conditionsapproaching those taking place naturally with the cry genes (low copynumber plasmid). Consequently, the 713 bp H₃-P₁ fragment was clonedbetween the HindIII and PstI sites of pHT304′lacZ to give pHT7901′lacZ.Finally, the H₂-H₃ fragment was cloned into the HindIII site ofpHT7901′lacZ to give pHT7902′lacZ which bears the H₂-H₃ fragment in itsoriginal orientation with respect to the H₃-P₁ fragment (FIG. 4). Theplasmids pHT7901′lacZ, pHT7902′lacZ and pHT7902′lacZ were introducedinto B. thuringiensis subspecies Kurstaki HD1 Cry⁻B by electroporation.The vector pHT304′lacZ had a blue phenotype potentially attributable tothe lacZ promoter or to another DNA region of pUC19 acting as promoter,located upstream from the cloning sites. The sporulation of each strainwas induced and samples were taken at t⁻² and t⁻¹ (2 hours and 1 hourbefore the triggering of sporulation, respectively) and at to t₀ tot_(7.5) at intervals of 1.5 hour and tested for beta-galactosidaseactivity (FIG. 5). The beta-galactosidase activity of the strain bearingpHT304′lacZ was constant at about 800 Miller units from t⁻² to t_(7.5).The level of the production of enzymes of the strain bearingpHT7901′lacZ rose from about 250 Miller units at t⁻² to about 1,200Miller units at t_(7.5), indicating a small but significant increase ofthe beta-galactosidase activity during sporulation (this increase is notapparent because of the scale used in FIG. 5). On the other hand, therecombinant strain bearing pHT7902′lacZ produced much beta-galactosidase(33,000 Miller units at t₆ and t_(7.5)). Its beta-galactosidase activityincreases from about 20 fold between t₀ and t₆ (FIG. 5). The ratio ofthe activities of the strains bearing pHT7901′lacZ and pHT7902′lacZincreased from 8 fold during the phase of vegetative growth to about 25fold during the late phase of sporulation.

[0157] The results presented above and more precisely the FIGS. 4 and 5indicate that the cryIIIA expression system is functional (at low copynumber) if the H₂-H₃ region is present upstream from the H₃-H₁ region.If this is the case, very high levels of expression are obtained whetherwith the cryIIIA gene or with the lacZ gene.

[0158] 1) Precise Definition of the Enhancer Region

[0159] Deletions from the H₂-P₁ fragment (FIG. 3A) showed that aTaqI-TaqI fragment (positions 907 to 1559, FIG. 3) was sufficient toobtain the strong expression of the lacZ gene (plasmid pHT7930′lacZ,FIG. 6).

[0160] Furthermore, an internal deletion from the fragment between thePacI and Xmn I sites (positions 990 to 1179) does not reduce theexpression of the lacZ gene.

[0161] This internal deletion led to the introduction of a linkerbetween the PacI and XmnI sites.

[0162] The following two nucleotides were synthesized and hybridizedtogether to construct a double-stranded DNA sequence capable of servingas linker between the PacI and XmnI sites: -5′-TAAAGATATCTTTGAAGCTTCACGTGTTTAAACAGGCCT GCAG -3′- -3′-TAATTTCTATAGAAACTTCGAAGTGCACAAATTTGTCCG GACGTC -5′-

[0163] The linker used here has a sequence such that five nucleotidesnaturally present after the PacI site are reconstituted in the plasmidpHT7931′lacZ.

[0164] In the presence of this deletion, a better expression seems to beobtained by bringing closer together the two regions TaqI-PacI(positions 907 to 990) and XmnI-TaqI (positions 1179 to 1559) (plasmidpHT7931′lacZ, FIG. 6).

[0165] It follows that the cryIIIA expression system requires theassociation of two distinct DNA sequences; one is included between theTaqI and PacI sites (positions 907 to 990), the other is includedbetween the XmnI and TaqI sites (positions 1179 to 1559).

[0166] This conclusion is reinforced by the fact that in the absence ofthe XmnI-TaqI region (positions 1179 to 1559), the region situatedupstream from the XmnI site is not sufficient to obtain the high levelof expression of the lacZ gene (plasmid pHT7907′lacZ, FIG. 6). In fact,the DraI-XmnI DNA sequence (positions 806 to 1179) placed upstream fromthe lacZ gene (plasmid pHT7907′lacZ makes it possible to obtain in Bt(B. thuringiensis) a beta-galactosidase activity of only about 3500Miller units (to be compared with 30,000 Mu obtained with the plasmidpHT7902′lacZ and pHT7903′lacZ).

[0167] Hence this result confirms that the association of the twosequences TaqI-PacI (positions 907 to 990) and XmnI-TaqI (positions 1179to 1559) is necessary in order for the cryIIIA expression system to befully functional.

[0168] The experiment performed with the DraI-XmnI fragment upstreamfrom lacZ (plasmid pHT7907′lacZ) indicates that a promoter activity isincluded between DraI and XmnI, and even between TaqI and PacI(positions 907 to 990) since the high beta-galactosidase activity isobtained when the PacI-XmnI fragment (positions 991 to 1179) is absent.

[0169] The analysis of the RNAs by primer extension carried out by usingan oligonucleotide complementary to the sequence included between thepositions 1090 and 1121 in fact makes it possible to detect aninitiation of transcription in this region. The latter is located inposition 983 (FIG. 3) or more probably at position 984. It follows fromthis that a promoter must be situated several base pairs upstream fromthis start. Although there is no obvious homology with known promoters,the −35 (TTGCAA) and −10 (TAAGCT) boxes of the promoter would beexpected to be found between the positions 945 to 980.

[0170] A MunI-PstI DNA fragment (positions 952 to 1612) placed in frontof lacZ (plasmid pHT7909′lacZ confers a weak beta-galactosidase activitycomparable to that obtained with the plasmid pHT7901′lacZ (FIGS. 4 and5).

[0171] This result suggests that the promoter situated at positions 945and 980 may be inactivated in a construction starting at MunI (position952).

[0172] However, it is known that the minimal sequence necessary for theexpression has been defined as starting at the TaqI site (position 907).

[0173] It follows from these different experiments that a DNA sequencelocated between the TaqI and PacI sites (positions 907 to 990) isrequired in order to obtain a high expression of lacZ and, consequently,a high level of transcription of cryIIIA.

Measurement of the Activity of the Upstream Promoter in the CryIIIAExpression System

[0174] In order to measure the activity of the upstream promoter, atranscriptional fusion was constructed with the DNA fragment containingthis promoter and the lacZ gene. For this the expression vectorpHT304-18Z was first constructed (FIG. 8A). The DNA fragment includedbetween the positions 907 and 990 was then cloned upstream of the lacZgene to give the plasmid pHT7832′lacZ. The beta-galactosidase activityis 3,000 U/ml of proteins at t₀ and 13,000 U/mg of proteins at t₆ (FIG.8B).

[0175] The role of the upstream promoter in the global activity of thecryIIIA expression system was evaluated by analyzing the effect producedby its inactivation. The MunI restriction site was filled in with theaid of the Klenow fragment of the DNA polymerase in the presence ofdeoxynucleotides to give the plasmid pHT7832ΔMunI′lacZ. This leads tothe addition of 4 nucleotides between the −35 and −10 regions of thepromoter (CAATTAATTG versus CAATTG). The beta-galactosidase activity ofthe strain bearing pHT7832ΔMunI′lacZ was about 10 U/mg of proteins at t₀and about 30 U/mg of proteins at t₆ (FIG. 8B). This result indicatesthat the upstream promoter is then inactivated. The DNA fragmentcontaining the modified MunI site was introduced into the plasmidpHT7830′lacZ to give the plasmid pHT7830ΔMunI′lacZ. Thebeta-galactosidase activity of the strain bearing pHT7830ΔMunI′lacZ wasabout 25 U/mg of proteins at t₀ and about 450 U/mg of proteins at t₆(FIG. 8B). By comparison with the strain bearing the plasmidpHT7830′lacZ, it follows that the upstream promoter is necessary for theoptimal functioning of the cryIIIA expression system. The plasmidpHT7830′lacZ corresponds to the vector pHT304-18Z in which is cloned theTaqI fragment containing the entire cryIIIA expression system.

Study of the Role of the Downstream Region in the CryIIIA ExpressionSystem

[0176] The preceding results confirm that the upstream promoter isnecessary for the optimal functioning of the cryIIIA expression system;on the other hand, it is not sufficient to account for the maximalactivity of the entire system. This latter aspect had been mentionedpreviously (compare the beta-galactosidase activity of the strainsbearing the plasmids pHT7832′lacZ and pHT7831′lacZ (FIG. 8B). Theplasmid pHT7831′lacZ corresponds to the plasmid pHT7830′lacZ, theinternal fragment PacI-XmnI of which is deleted. It follows that aregion called “downstream” is required to explain the maximal activityof the cryIIIA expression system.

[0177] The transcription initiation site of the cryIIIA gene had beenpreviously localized in position 1413, the −35 and −10 regions of theputative promoter ought to be included between the nucleotides 1370 and1412 (Sekar et al., 1987, Proc. Natl. Acad. Sci. USA, 84: 7036-7040). Inorder to assess the efficacy of this putative promoter, we haveconstructed the plasmid pHT7815/8′lacZ in which the DNA fragmentincluded between the nucleotides 1352 and 1412 was deleted. Thebeta-galactosidase activity of the strain bearing pHT7815/8′lacZ wasabout 3,000 U/mg of proteins at t₀ and about 42,000 U/mg of proteins att₆ (FIG. 8B). This result indicates that the region included between thenucleotides 1362 and 1412 does not play an essential role in the cryIIIAexpression system and can not therefore be considered as the promoter ofthe cryIIIA gene.

[0178] A primer extension experiment was carried out with the total RNAsextracted at t₃ from a B. thuringiensis strain bearing the plasmidpHT7815/8∝lacZ The 5′ end of the major transcript is detected aspreviously at position 1413 (FIG. 9). All of our results thusdemonstrate that this end does not correspond to transcriptioninitiation but to the end of a stable transcript initiated at position984 starting from a upstream promoter localized in the DNA regionincluded between the TaqI and PacI sites (positions 907 to 990) anddefined by the −35 and −10 regions: TTGCAA and TAAGCT. Since the 5′ endof the major cryIIIA transcript is invariably in position 1413, in thepresence or in the absence of the DNA fragment included between thepositions 1362 and 1412, it follows that this end is defined by thepresence of a DNA sequence which is found downstream of the position1413. The role of this region is thus exerted at thepost-transcriptional level. The analysis of this downstream sequence wasmade in B. subtilis with the aid of transcriptional fusions with thelacZ gene. The various constructions presented in FIG. 10 have enabledus to define more precisely the downstream region and to measure itspost-transcriptional effect:

[0179] 1. The DNA fragment included between the nucleotides 1462 and1556 was deleted from the plasmid pHT7830′lacZ to give the plasmidpHT7816′lacZ. The beta-galactosidase activity of the strain bearingpHT7816′lacZ was about 25,000 U/mg of proteins at t₃ whereas thebeta-galactosidase activity of the strain bearing pHT7830′lacZ was about50,000 U/mg of proteins at t₃ (FIG. 10).

[0180] 2. The DNA fragment included between the nucleotides 1413 and1556 was deleted from the plasmid pHT7830′lacZ to give the plasmidpHT7805′lacZ. The beta-galactosidase activity of the strain bearingpHT7805′lacZ was about 5,000 U/mg of proteins at t₃ (FIG. 10).

[0181] 3. The nucleotides GGA in position 1421-1423 of the plasmidpHT7830′lacZ were replaced by the nucleotides CCC to give the plasmidpHT7830Rm′lacZ. The beta-galactosidase activity of the strain bearingpHT7830Rm′lacZ was about 5,000 U/mg of proteins at t₃ (FIG. 10).

[0182] 4. A primer extension experiment was carried out with the totalRNAs extracted at t₃ from a B. thuringiensis strain bearing the plasmidpHT7830Rm′lacZ. The 5′ end of the major transcript is detected atposition 984 and no transcript having a 5′ end at position 1413 isdetected.

[0183] These four results indicate that the post-transcriptional effectof the downstream region is principally due to the nucleotide sequenceincluded between the nucleotides 1413 and 1461. Furthermore, thenucleotides GGA in position 1421-1423 are important for conferring thepost-transcriptional effect and might be modified only by consideringreplacement by a sequence ensuring an intensity of interaction with the16S ribosomal RNA similar to the intensity of interaction measured forthe nucleotides GGA. For example, the replacement of the nucleotides GGAby the nucleotides CCC leads to the complete disappearance of thepost-transcriptional effect, explained by a considerable modification ofthe intensity of interaction between this portion of the segment and the16S RNA. The downstream region thus defined has as distinctivecharacteristic that of containing a nucleotide sequence complementary tothe 3′ end of the 16S RNA of ribosome.

[0184] The post-transcriptional effect of this DNA sequence has thenbeen evaluated by using a heterologous expression system: the followingDNA sequence (S1): 5′-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3′3′-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′

[0185] was synthesized and cloned between the HindIII and PstI sites ofthe vector pHT304′lacZ to give the plasmid pHT304ΠRS1′lacZ. This DNAsequence is thus intercalated between the promoter of the lacZ gene andthe sequence coding forthe lacZ gene. The beta-galactosidase activity ofthe strain 168 of B. subtilis bearing pHT304ΠRS1′lacZ was about 4,000U/mg of proteins at t₃. It follows that the sequence described aboveincreases by a factor of 4 the expression of the lacZ gene. Thisincrease is comparable to the increase due to the region includedbetween the nucleotides 1413 and 1461, i.e by a factor of 5 (compare thebeta-galactosidase activity of the B. subtilis strains containing theplasmids pHT7816′lacZ or pHT7805′lacZ). The following DNA region is thussufficient to confer the post-transcriptional effect to the cryIIIAexpression system: 5′-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3′3′-GAACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′

[0186] This sequence posseses a region complementary to the 3′ end ofthe 16S ribosomal RNA. However, other elements characteristic of thedownstream region of the cryIIIA expression system and which mayaccentuate this effect, in particular by preventing the movement of theribosome, are probably comprised in the nucleotide sequence includedbetween positions 1462 and 1556. Their presence seems to explain thedifference of beta-galactosidase activity observed between the B.subtilis strain containing the plasmid pHT7830′lacZ (50,000 U/mg ofproteins at t₃) and the B. subtilis strain containing the plasmidpHT7816′lacZ (25,000 U/mg of proteins at t₃; see FIG. 10).

[0187] These results thus seem to confirm that the post-transcriptionaleffect of the downstream region results from the hybridization betweenthe 16S ribosomal RNA and the S2 sequence of the messenger RNA ofcryIIIA. It is hence probable that the ribosome or a part of theribosome binds to this downstream region of the RNA and thus protects itfrom exonucleolytic degradation initiated at 5′. As previouslymentioned, this binding would thus have the effect of enhancing thestability of the messengers and thus of increasing the level ofexpression of a given gene. That explains why the 5′ end of the cryIIIAtranscripts is invariably at position 1413 irrespective of wheretranscription is initiated. This mechanism also seems to be confirmed bythe positive effect of the S1 sequence on a heterologous expressionsystem (plasmid pHT304′ΠRS1lacZ in the strain 168 of B. subtilis).

Introduction of the Fusion {CryIIIA-LacZ Expression System} into theChromosome of Bacillus subtilis

[0188] The vector pAF1, non-replicative in B. subtilis enables thefusions with the LacZ reporter gene to be introduced into the B.subtilis chromosome at the amyE locus (J. Bact. 1990, 172: 835-844). Theplasmid pHC1 is obtained by insertion of the HindIII-SacI fragment (2.7kb) of the pHT7901′LacZ between the HindIII-SacI sites of pAF1.

[0189] The plasmid pHC2 is obtained by insertion of the HindIII-SacIfragment (3.7 kb) of the pHT7902′LacZ between the HindIII and SacI sitesof pAF1.

[0190] The fusions are introduced into the B. subtilis strain 168 trpC2(Anagnostopoulos, C and Spizizen, J. 1961 J. Bacteriol. 81: 741-746)(Bacillus subtilis 168) by transformation; the {amy-} phenotype accountsfor the integration by double recombination.

Study of the Expression System of the CryIIIA Gene in B. subtilis

[0191] The B. subtilis strains obtained after transformation andintegration of the pHC1 and pHC2 plasmids are called respectively:

[0192] Bs168 {H} and Bs168 {P}

[0193] The construction contained in the plasmid pHC2, i.e bearing theH₂-P₁ fragment upstream from the lacZ, was also introduced into the B.subtilis strain Δ sigE.

[0194] The strain ΔsigE is obtained by transforming a parental strain(Spo⁺) with a plasmid non-replicative in Gram-positive bacteria andbearing a sigE gene, the internal region of which is deleted. The sigEgene was described by Stragier et al 1984 Nature 312: 376-378.

[0195] The strain Δ sigE is transformed with the plasmid pHC2 and theresulting strain is Δ sigE {P}.

[0196] The gene coding for the sigmaE factor specific for sporulationhas been deleted from this strain. This strain is hence asporogenic(Spo⁻).

[0197] Similarly, the strain Bs 168 {P} was transformed with a “Km^(R)cassette” which interrupts the SpoOA gene. The strain in which the SpoOAgene interrupted by a “KmR cassette” originates is obtained bytransforming a parental strain (Spo⁺) with a plasmid, non-replicative inGram-positive bacteria and bearing a SpoOA gene (described by Ferrari,F. A. et al. 1985 PNAS USA 82: 2647-2651) interrupted by a gene forresistance to kanamycin. The chromosomal DNA of this strain was used totransform the strain Bs 168 {P}.

[0198] Thus, the resulting Spo-strain was called Bs 168 SpoOA {P}.

[0199] Firstly, it appears that the production of beta-galactosidaseobtained with the strain of B. subtilis 168 {H} is very low (<100 μM) bycomparison with the strain 168 {P} (about 15,000 μM). These results aresimilar to those obtained in Bt.

[0200] Furthermore, a very surprising result was obtained: theexpression in the strain BsΔsigE is identical with the expression in thewild type strain Bs 168. This result indicates that the cryIIIA gene isnot controlled by a specific promoter of the sigma E factor as is thecase for the cryIA gene.

[0201] It is even more surprising that the expression in the strain BsSpoOA {P} is higher than that obtained in the strain Bs 168 {P}. Thisresult shows that the expression of cryIIIA is independent ofsporulation since the spoOA gene is implicated in the first stage ofsporulation.

[0202] These results are very important for the development and theapplications of the cryIIIA expression system. They in fact indicatethat it is possible to envisage the production of the insecticidaltoxins or of any other protein of commercial interest in Spo⁻ strains ofB. subtilis or B. thuringiensis.

Analysis of the Expression of the Fusion {CryIIIA-LacZ ExpressionSystem=pHC2} in Bacillus subtilis as a Function of the Culture Medium

[0203] It is possible to make the following observations as regards theexpression of the fusion in the media 1 to 5, respectively, thecomposition of which is given below,

[0204] Expression (although weak) occurs during the vegetative phase.

[0205] Expression increases at the beginning of the stationary phase.

[0206] The comparison of media 2 (deficient in phosphate) and 5(deficient in amino acids) show that the CryIIIA expression system isactivated by the amino acids deficency.

[0207] The expression in medium 4 shows that this activation requiresthe presence of salts: CaCl₂, MnCl₂, AFC

[0208] The activation is independent of sporulation:

[0209] In sporulation medium 1 (Sp medium) expression stops at t₂.

[0210] In the medium 5 the cells cannot sporulate (glucose inhibitssporulation) and activation is maximum.

[0211] When the only nitrogen source is NH⁺ ₄, the activation is lower,expression, however, remains considerable (medium 3).

[0212] 1/Sp Medium: sporulation medium

[0213] 8 g nutrient broth (Difco)/liter

[0214] 1 mM MgSO₄

[0215] 13 mM KCl

[0216] 10 μM MnCl₂

[0217] 1 μM FeSO₄

[0218] 1 mM CaCl₂

[0219]2/Phosphate Deficient Medium

[0220] HEPES buffer pH 7; 50 mM

[0221] 1 mM MgSO₄

[0222] 0.5 mM CaCl₂

[0223] 10 M MnCl₂

[0224] 4.4 mg/liter ammonium ferric citrate (AFC)

[0225] 2% glucose

[0226] 10mM KCl

[0227] 100 mg/liter of each amino acid

[0228] 50 mg/liter tryptophan

[0229] 0.45 mM phosphate buffer, pH 7

[0230] 3/Minimal Medium

[0231] 44 mM KH₂PO₄

[0232] 60 mM K₂HPO₄

[0233] 2.9 mM Trisodium citrate

[0234] 15 mM (NH₄)₂SO₄

[0235] 2% glucose

[0236] 4/Amino Acid Deficient Medium Without CaCl₂, MnCl₂, AFC

[0237] 44 mM KH₂PO₄

[0238] 60 mM K₂HPO₄

[0239] 2.9 mM Trisodium citrate

[0240] 2% glucose

[0241] 1 mM MgSO₄

[0242] 50 mg/liter tryptophan

[0243] 0.5 casein hydrolysate (CH)

[0244] 5/4 Idem by Adding:

[0245] 0.5 mM CaCl₂

[0246] 10 M MnCl₂

[0247] 4.4 mg/liter AFC

Construction of a B. thuringiensis Sp⁻ Strain

[0248] Cloning of the spoOA Gene of B. thuringiensis:

[0249] The total DNA of the B. thuringiensis strain 407 of serotype 1was purified and digested by the enzyme HindIII. The HindIII fragmentswere ligated with the vector pHT304 digested by HindIII and the ligationmixture was used to transform the B. subtilis strain 168. Thetransformant clones were selected for resistance to erythromycin. Theywere then transformed with the total DNA of the B. subtilis strain 168,the spoOA gene of which was interrupted by a “Km^(R) cassette”. Thetransformant clones which had become resistant to kanamycin which stillhad a Spo⁺ phenotype were studied. One of the clones carried arecombinant plasmid capable of compensating the spoOA mutation of B.subtilis. This plasmid was constituted by the vector pHT304 and aHindIII fragment of about 2.4 kb (FIG. 11A).

[0250] Determination of the Nucleotide Sequence of the SpoOA Gene of B.thuringiensis:

[0251] The nucleotide sequence of the HindIII fragment was determinedand revealed the presence of an open reading frame of 804 bp capable ofcoding for a protein of 264 amino acids homologous to the SpoOA proteinof B. subtilis. The nucleotide sequence of 804 bp of the spoOA gene ofB. thuringiensis strain 407 is shown in FIG. 11B.

[0252] Interruption of the SpoOA Gene of B. thuringiensis:

[0253] A 1.5 kb DNA fragment bearing an aphIII gene, conferringresistance of kanamycin (“cassette Km^(R)”), was inserted between thetwo HincII sites of the spoOA gene (FIG. 11) A 40 bp fragment includedbetween the positions 267 and 307 of the spoOA gene was thus replaced bythe “Km^(R) cassette”. The HindIII DNA fragment of about 3.9 kbcontaining the spoOA gene interrupted by the “Km^(R) cassette” wascloned in the thermosensitive vector pRN5101 (Villafane et al. 1987, J.Bacteriol. 169: 4822-4829). The resulting plasmid (designated pHT5120)was introduced in the B. thuringiensis strain 407 Cry⁻ byelectroporation. The spoOA gene of the B. thuringiensis strain 407 Cry⁻was replaced by the copy interrupted with the “Km^(R) cassette” bygenetic recombination in vivo by using the protocol previously described(Lereclus et al., 1992, Bio/Technology 10: 418-421). The resultant B.thuringiensis strain (designated 407-OA::KmR) is resistant to kanamycin(300 μg/ml) and does not produce spores when it is cultured in HCTmedium, usually favorable to the sporulation of B. thuringiensis. ADNA/DNA hybridization experiment performed with the 2.4 kb HindIIIfragment as probe revealed that the spoOA gene of the B. thuringiensisstrain 407 Cry⁻ has indeed been replaced by the copy interrupted withthe “Km^(R) cassette”.

[0254] Production of the CryIIIA Toxin in the B. thuringiensis Strain407-OA::Km^(R):

[0255] The plasmid pHT305P bearing the cryIIIA gene was introduced intothe B. thuringiensis strain 407-OA::KmR by electroporation. Therecombinant clone obtained was deposited with the CNCM on Mar. 5, 1994and to which the access number I-1412 was assigned. The recombinantclone obtained was cultured at 30° C. in HCT medium+glucose 3 g/l or inLB medium (NaCl, 5 g/l; yeast extract, 5 g/l; Bacto tryptone 10 g/l) toestimate the production of toxins. After about 48 hours the bacteriacontained a crystal visible by examination with the optical microscope.This crystal was rhomboidal, characteristic of the crystals constitutedby the CryIIIA protein. The crystals produced by the B. thuringiensisstrain 407-OA::KmR {pHT315} are of considerable size and remain includedin the cells several days after the latter have ceased to develop in HCTmedium; in LB medium a portion of the cells lyse and the crystals arereleased. The crystals are constituted of proteins of about 70 kDa(CryIIIA) specifically toxic for the Coleoptera.

1 17 1692 base pairs nucleic acid single linear DNA (genomic) 1AAGCTTTCAG TGAAGTACGT GATTATACGG AGATGAAAAT TCGTACACTG TTAACGAGAA 60GGAAACGCCG ACGAAAGCGT AGCATCGGAT GGCAAAGATG GAGTAACGAA TATCTCTACG 120GTGTACTGGG GCTTTACTGA GACTAGAAAG TCCTTCCCTT GAAAAGTGCA GAGAGTTTTC 180GATAAAAGTG TCAGCCATTT GATAAGTCTC ATTCTCATAA CCTATTGATG AAGTTTATAG 240GGAAGCTGCT TGAGAGGGAA AACCTCACGA ACAGTTCTTA TGGGGAGAGA CTGGAAACAG 300GTCACAATTG ATACCTCGCT AATCTTTTAA CCGACAAAGT TTTTTTAAAC CGTGGAAGTC 360ATAATAACCT GGATATTGTG AATTTATAAA AGTTAACAAA TGGTTTATAT TAAGACAGTC 420ATAAACCAAA GATTTTTCTT CTAAAGCTAC GATAGCAAAA ATTTCACTAG AAATTAGTTA 480TACAAGCATT TTGTAAGAAT TATTAAAAAG ATAAATCCTG CTATTACGAG ATTAGTAGGA 540TGATATTGTG AAAAATTTTT TATCTATTCG ATTTAAAATA TTTATGAATT TTACATAAAC 600CTCATAAGAA AAAATACTAT CTATACTATT TTAAGAAATT TATTAGAATA AGCGGATTCA 660AAATAGCCCT GGCCATAAAA GTACCTCAGC AGTAGAAGTT TTGACCAAAA TTAAAAAAAT 720ACCCAATCAA GAGAATATTC TTAATTACAA TACGTTTTGC GAGGAACATA TTGATTGAAA 780TTTAATAAAT TTAGTCCTAA AATTTAAAGA AATTTAAGTT TTTCATATTT TTATGAACTA 840ACAAGAATAA AAATTGTGTT TATTTATTAT TCTTGTTAAA TATTTGATAA AGAGATATAT 900TTTTGGTCGA AACGTAAGAT GAAACCTTAG ATAAAAGTGC TTTTTTTGTT GCAATTGAAG 960AATTATTAAT GTTAAGCTTA ATTAAAGATA ATATCTTTGA ATTGTAACGC CCCTCAAAAG 1020TAAGAACTAC AAAAAAAGAA TACGTTATAT AGAAATATGT TTGAACCTTC TTCAGATTAC 1080AAATATATTC GGACGGACTC TACCTCAAAT GCTTATCTAA CTATAGAATG ACATACAAGC 1140ACAACCTTGA AAATTTGAAA ATATAACTAC CAATGAACTT GTTCATGTGA ATTATCGCTG 1200TATTTAATTT TCTCAATTCA ATATATAATA TGCCAATACA TTGTTACAAG TAGAAATTAA 1260GACACCCTTG ATAGCCTTAC TATACCTAAC ATGATGTAGT ATTAAATGAA TATGTAAATA 1320TATTTATGAT AAGAAGCGAC TTATTTATAA TCATTACATA TTTTTCTATT GGAATGATTA 1380AGATTCCAAT AGAATAGTGT ATAAATTATT TATCTTGAAA GGAGGGATGC CTAAAAACGA 1440AGAACATTAA AAACATATAT TTGCACCGTC TAATGGATTT ATGAAAAATC ATTTTATCAG 1500TTTGAAAATT ATGTATTATG ATAAGAAAGG GAGGAAGAAA AATGAATCCG AACAATCGAA 1560GTGAACATGA TACAATAAAA ACTACTGAAA ATAATGAGGT GCCAACTAAC CATGTTCAAT 1620ATCCTTTAGC GGAAACTCCA AATCCAACAC TAGAAGATTT AAATTATAAA GAGTTTTTAA 1680GAATGACTGC AG 1692 653 base pairs nucleic acid single linear DNA(genomic) misc_feature 1..653 /note= “NUCLEOTIDES 907 TO 1559 OF SEQ IDNO1” 2 TCGAAACGTA AGATGAAACC TTAGATAAAA GTGCTTTTTT TGTTGCAATT GAAGAATTAT60 TAATGTTAAG CTTAATTAAA GATAATATCT TTGAATTGTA ACGCCCCTCA AAAGTAAGAA 120CTACAAAAAA AGAATACGTT ATATAGAAAT ATGTTTGAAC CTTCTTCAGA TTACAAATAT 180ATTCGGACGG ACTCTACCTC AAATGCTTAT CTAACTATAG AATGACATAC AAGCACAACC 240TTGAAAATTT GAAAATATAA CTACCAATGA ACTTGTTCAT GTGAATTATC GCTGTATTTA 300ATTTTCTCAA TTCAATATAT AATATGCCAA TACATTGTTA CAAGTAGAAA TTAAGACACC 360CTTGATAGCC TTACTATACC TAACATGATG TAGTATTAAA TGAATATGTA AATATATTTA 420TGATAAGAAG CGACTTATTT ATAATCATTA CATATTTTTC TATTGGAATG ATTAAGATTC 480CAATAGAATA GTGTATAAAT TATTTATCTT GAAAGGAGGG ATGCCTAAAA ACGAAGAACA 540TTAAAAACAT ATATTTGCAC CGTCTAATGG ATTTATGAAA AATCATTTTA TCAGTTTGAA 600AATTATGTAT TATGATAAGA AAGGGAGGAA GAAAAATGAA TCCGAACAAT CGA 653 84 basepairs nucleic acid single linear DNA (genomic) misc_feature 1..84 /note=“NUCLEOTIDES 907 TO 990 OF SEQ ID NO1” 3 TCGAAACGTA AGATGAAACCTTAGATAAAA GTGCTTTTTT TGTTGCAATT GAAGAATTAT 60 TAATGTTAAG CTTAATTAAAGATA 84 381 base pairs nucleic acid single linear DNA (genomic)misc_feature 1..381 /note= “NUCLEOTIDES 1179 TO 1559 OF SEQ ID NO1” 4TTGTTCATGT GAATTATCGC TGTATTTAAT TTTCTCAATT CAATATATAA TATGCCAATA 60CATTGTTACA AGTAGAAATT AAGACACCCT TGATAGCCTT ACTATACCTA ACATGATGTA 120GTATTAAATG AATATGTAAA TATATTTATG ATAAGAAGCG ACTTATTTAT AATCATTACA 180TATTTTTCTA TTGGAATGAT TAAGATTCCA ATAGAATAGT GTATAAATTA TTTATCTTGA 240AAGGAGGGAT GCCTAAAAAC GAAGAACATT AAAAACATAT ATTTGCACCG TCTAATGGAT 300TTATGAAAAA TCATTTTATC AGTTTGAAAA TTATGTATTA TGATAAGAAA GGGAGGAAGA 360AAAATGAATC CGAACAATCG A 381 378 base pairs nucleic acid single linearDNA (genomic) misc_feature 1..378 /note= “NUCLEOTIDES 1179 TO 1556 OFSEQ ID NO1” 5 TTGTTCATGT GAATTATCGC TGTATTTAAT TTTCTCAATT CAATATATAATATGCCAATA 60 CATTGTTACA AGTAGAAATT AAGACACCCT TGATAGCCTT ACTATACCTAACATGATGTA 120 GTATTAAATG AATATGTAAA TATATTTATG ATAAGAAGCG ACTTATTTATAATCATTACA 180 TATTTTTCTA TTGGAATGAT TAAGATTCCA ATAGAATAGT GTATAAATTATTTATCTTGA 240 AAGGAGGGAT GCCTAAAAAC GAAGAACATT AAAAACATAT ATTTGCACCGTCTAATGGAT 300 TTATGAAAAA TCATTTTATC AGTTTGAAAA TTATGTATTA TGATAAGAAAGGGAGGAAGA 360 AAAATGAATC CGAACAAT 378 591 base pairs nucleic acidsingle linear DNA (genomic) misc_feature 1..447 /note= “NUCLEOTIDES 1 TO447 CORRESPOND TO NUCLEOTIDES 907 TO 1353 OF SEQ ID NO1” misc_feature448..591 /note= “NUCLEOTIDES 448 TO 591 CORRESPOND TO NUCLEOTIDES 1413TO 1556 OF SEQ ID NO1” 6 TCGAAACGTA AGATGAAACC TTAGATAAAA GTGCTTTTTTTGTTGCAATT GAAGAATTAT 60 TAATGTTAAG CTTAATTAAA GATAATATCT TTGAATTGTAACGCCCCTCA AAAGTAAGAA 120 CTACAAAAAA AGAATACGTT ATATAGAAAT ATGTTTGAACCTTCTTCAGA TTACAAATAT 180 ATTCGGACGG ACTCTACCTC AAATGCTTAT CTAACTATAGAATGACATAC AAGCACAACC 240 TTGAAAATTT GAAAATATAA CTACCAATGA ACTTGTTCATGTGAATTATC GCTGTATTTA 300 ATTTTCTCAA TTCAATATAT AATATGCCAA TACATTGTTACAAGTAGAAA TTAAGACACC 360 CTTGATAGCC TTACTATACC TAACATGATG TAGTATTAAATGAATATGTA AATATATTTA 420 TGATAAGAAG CGACTTATTT ATAATCATCT TGAAAGGAGGGATGCCTAAA AACGAAGAAC 480 ATTAAAAACA TATATTTGCA CCGTCTAATG GATTTATGAAAAATCATTTT ATCAGTTTGA 540 AAATTATGTA TTATGATAAG AAAGGGAGGA AGAAAAATGAATCCGAACAA T 591 465 base pairs nucleic acid single linear DNA (genomic)misc_feature 1..84 /note= “NUCLEOTIDES 1 TO 84 CORRESPOND TO NUCLEOTIDES907 TO 990 OF SEQ ID NO1” misc_feature 85..465 /note= “NUCLEOTIDES 85 TO465 CORRESPOND TO NUCLEOTIDES 1179 TO 1559 OF SEQ ID NO1” 7 TCGAAACGTAAGATGAAACC TTAGATAAAA GTGCTTTTTT TGTTGCAATT GAAGAATTAT 60 TAATGTTAAGCTTAATTAAA GATATTGTTC ATGTGAATTA TCGCTGTATT TAATTTTCTC 120 AATTCAATATATAATATGCC AATACATTGT TACAAGTAGA AATTAAGACA CCCTTGATAG 180 CCTTACTATACCTAACATGA TGTAGTATTA AATGAATATG TAAATATATT TATGATAAGA 240 AGCGACTTATTTATAATCAT TACATATTTT TCTATTGGAA TGATTAAGAT TCCAATAGAA 300 TAGTGTATAAATTATTTATC TTGAAAGGAG GGATGCCTAA AAACGAAGAA CATTAAAAAC 360 ATATATTTGCACCGTCTAAT GGATTTATGA AAAATCATTT TATCAGTTTG AAAATTATGT 420 ATTATGATAAGAAAGGGAGG AAGAAAAATG AATCCGAACA ATCGA 465 49 base pairs nucleic acidsingle linear DNA (genomic) misc_feature 1..49 /note= “CORRESPONDS WITHNUCLEOTIDES 1413 TO 1461 OF SEQ ID NO1” 8 TCTTGAAAGG AGGGATGCCTAAAAACGAAG AACATTAAAA ACATATATT 49 38 base pairs nucleic acid doublelinear DNA (genomic) 9 AGCTTGAAAG GAGGGATGCC TAAAAACGAA GAACTGCA 38 32base pairs nucleic acid double linear DNA (genomic) 10 CTTGAAAGGAGGGATGCCTA AAAACGAAGA AC 32 144 base pairs nucleic acid single linearDNA (genomic) misc_feature 1..144 /note= “CORRESPONDS TO NUCLEOIDES 1413TO 1556 OF SEQ ID NO1” 11 TCTTGAAAGG AGGGATGCCT AAAAACGAAG AACATTAAAAACATATATTT GCACCGTCTA 60 ATGGATTTAT GAAAAATCAT TTTATCAGTT TGAAAATTATGTATTATGAT AAGAAAGGGA 120 GGAAGAAAAA TGAATCCGAA CAAT 144 28 base pairsnucleic acid single linear DNA (genomic) 12 CGTAATCTTA CGTCAGTAACTTCCACAG 28 39 base pairs nucleic acid single linear DNA (genomic) 13CTTAGGCTTG TTAGCTTCAC TTGTACTATG TTATTTTTG 39 32 base pairs nucleic acidsingle linear DNA (genomic) 14 GTTAGATAAG CATTTGAGGT AGAGTCCGTC CG 32 88base pairs nucleic acid double linear DNA (genomic) 15 TAAAGATATCTTTGAAGCTT CACGTGTTTA AACAGGCCTG CAGTAATTTC TATAGAAACT 60 TCGAAGTGCACAAATTTGTC CGGACGTC 88 804 base pairs nucleic acid single linear DNA(genomic) 16 GGAGGAAAAG CTGTGGAGAA AATTAAAGTA TGTCTTGTGG ATGATAATAAAGAATTAGTA 60 TCAATGTTAG AGAGCTATGT AGCCGCCCAA GATGATATGG AAGTAATCGGTACTGCTTAT 120 AATGGTCAAG AGTGTTTAAA CTTATTAACA GATAAGCAAC CTGATGTACTCGTTTTAGAC 180 ATTATTATGC CACACTTAGA TGGTTTAGCT GTATTGGAAA AAATGCGACATATTGAAAGG 240 TTAAAACAGC CTAGCGTAAT TATGTTGACA GCATTCGGGC AAGAAGATGTGACGAAAAAA 300 GCAGTTGACT TAGGTGCCTC GTATTTCATA TTAAAACCAT TTGATATGGAGAATTTAACG 360 AGTCATATTC GTCAAGTGAG TGGTAAAGCA AACGCTATGA TTAAGCGTCCACTACCATCA 420 TTCCGATCAG CAACAACAGT AGATGGAAAA CCGAAAAACT TAGATGCGAGTATTACGAGT 480 ATCATTCATG AAATTGGTGT ACCCGCTCAT ATTAAAGGAT ATATGTATTTACGAGAAGCA 540 ATCTCCATGG TATACAATGA TATCGAATTA TTAGGATCGA TTACGAAAGTATTGTATCCA 600 GATATCGCAA AGAAATATAA TACAACAGCC AGCCGTGTGG AGCGCGCAATTCGTCACGCA 660 ATTGAAGTAG CTTGGAGCCG TGGGAATATT GATTCTATTT CGTCCTTATTCGGTTATACA 720 GTATCCATGT CAAAAGCAAA ACCTACGAAC TCTGAGTTTA TCGCAATGGTTGCGGATAAG 780 CTGAGACTTG AACATAAAGC TAGT 804 10 base pairs nucleic aciddouble linear DNA (genomic) 17 CAATTAATTG 10

1. DNA sequence for the control of the expression of a sequence codingfor a gene in a host cell, said sequence being characterized in that itcomprises a promoter and a nucleotide sequence or downstream regionunder the control of said promoter, and in particular situateddownstream from said promoter and upstream from said coding sequence,said nucleotide sequence or downstream region containing a regionessentially complementary to the 3′ end of a bacterial ribosomal RNA,said DNA sequence being capable of intervening in order to increase theexpression of said coding sequence, placed downstream in a cell host. 2.DNA sequence according to claim 1, characterized by the followingproperties: it is included in a DNA sequence about 1692 bp long, definedby the restriction sites HindIII-PstI (H₂-P₁ fragment) such as obtainedby partial digestion of the 6 kb BamHI fragment borne by the cryIIIAgene of Bacillus thuringiensis strain LM79; it is capable of interveningin the control of the expression of a coding nucleotide sequence placeddownstream in a bacterial cell host, in particular of the Bacillusthuringiensis and/or Bacillus subtilis type.
 3. DNA sequence accordingto claim 2 characterized in that it corresponds to the HindIII-PstIsequence about 1692 bp long (H₂-P₁ fragment) such as that obtained bypartial digestion of the 6 kb BamHI fragment bearing the cryIIIA gene ofBacillus thuringiensis strain LM79.
 4. DNA sequence according to claim1, characterized in that it corresponds to the following nucleotidesequence designated by the expression Seq. No.1 included betweennucleotides 1 and 1692 of the sequence shown in FIG.
 3. 5. DNA sequenceaccording to any one of the claims 2 to 4, characterized in that itcorresponds to the sequence defined by the restriction site TaqI-TaqI.6. DNA sequence according to any one of the claims 1 to 5, characterizedin that it corresponds to the following nucleotide sequence designatedby the expression Seq No.2 included between nucleotides 907 and 1559 ofthe sequence shown in FIG.
 3. 7. DNA sequence according to any one ofthe claims 2 to 4, characterized in that it comprises the TaqI-PacIfragment corresponding to the sequence Seq No.3, in combination with theXmnI-TaqI fragment corresponding to sequence Seq No.4.
 8. DNA sequenceaccording to any one of the claims 2 to 7, characterized in that it is asequence intervening in the control of the transcription of the codingnucleotide sequence or a sequence intervening to increase the expressionof a coding sequence.
 9. DNA sequence according to claims 1 to 18,characterized by: its capacity to hybridize under non-stringentconditions with a nucleotide sequence selected from the sequences SeqNo.1, Seq. No.2 and Seq. No.3 and Seq. No.4 and by its capacity tointervene in the control of the expression in a cell host, in particularof the Bacillus type, of a coding nucleotide sequence placed downstream.10. Sequence according to claim 1, characterized in that said nucleotidesequence or downstream region is the sequence S2 isolated from abacterium, in particular from a Gram⁺ bacterium, more particularly froma bacterium of the Bacillus type, or a sequence derived from saidsequence S2, said S2 sequence being characterized in that it contains aregion essentially complementary to the 3′ end of the 16S RNA of abacterial ribosome, it is capable of intervening at thepost-transcriptional level when said coding sequence is expressed, inparticular without acting directly on the translation of the codingsequence to be expressed, in particular when said S2 sequence issituated between said promoter and said coding sequence for a gene so asto enhance the expression of said coding sequence in a bacterium, inparticular a bacterium of the Bacillus type.
 11. DNA sequence accordingto claim 10, characterized in that said sequence S2 contains a firstregion sufficiently complementary to the 3′ end of the 16S RNA of abacterial ribosome, in particular of a Gram⁺ bacterium, moreparticularly of a bacterium of the Bacillus type so that said firstregion is capable of hybridizing to a bacterial ribosome or to a part ofsaid ribosome, and a second region downstream from the first region,said second region containing a sequence capable of having an additionalpositive effect at the level of the expression of the coding sequence.12. DNA sequence according to claim 10, characterized in that thenucleotide sequence or the downstream region exhibits the followingproperties: it is contained in a nucleotide sequence hybridizing undernon-stringent conditions with the DNA fragment included between thenucleotides 1413 and 1559 of the sequence shown in FIG. 3; it is capableof intervening in the control of the expression of the coding sequenceof a gene in a host cell, in particular of a sequence coding forBacillus polypeptide toxic towards insects or a sequence coding for apolypeptide expressed during the vegetative phase, during the stationaryphase or during the sporulation of the Bacillus.
 13. DNA sequenceaccording to any one of the claims 2 to 12, characterized in that thenucleotide sequence or the downstream region corresponds to the sequencedefined by the XmnI-TaqI restriction sites.
 14. DNA sequence accordingto claim 13, characterized in that the nucleotide sequence or downstreamregion corresponds to the sequence Seq. No.4 included between thenucleotides 1179 and 1559 of the sequence shown in FIG. 3, or in that itcorresponds to any part of at least ten nucleotides of this sequence,naturally consecutive or not, capable of intervening in the control ofthe expression of a coding nucleotide sequence placed downstream in acell host.
 15. DNA sequence according to claim 13, characterized in thatthe nucleotide sequence or the downstream region corresponds to thesequence Seq No.5 corresponding to the fragment comprising thenucleotides 1179 to 1556 of the sequence shown in FIG.
 3. 16. DNAsequence according to claim 13, characterized in that the nucleotidesequence or the downstream region corresponds to the sequence Seq No.11corresponding to the fragment comprising the nucleotides 1413 to 1556 ofthe sequence shown in FIG.
 3. 17. DNA sequence according to claim 13,characterized in that the nucleotide sequence or the downstream regioncorresponds to the sequence Seq No.8 corresponding to the fragmentcomprising the nucleotides 1413 to 1461 of the sequence shown in FIG. 3.18. DNA sequence according to claim 13, characterized in that thenucleotide sequence or the downstream region corresponds to the sequenceSeq No.9 corresponding to the following DNA fragment5′-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3′3′-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′


19. Sequence according to claim 13, characterized in that the nucleotidesequence or downstream region corresponds to the sequence Seq No.10corresponding to the following DNA fragment5′-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3′3′-GAACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′


20. Sequence according to claim 13, characterized in that the nucleotidesequence or downstream region corresponds to the following DNA fragment:5′-GAAAGGAGG-3′ 3′-CTTTCCTCC-5′


21. DNA sequence according to any one of the claims 1 to 20,characterized in that the promoter is included in the sequence definedby the TaqI-PacI restriction sites.
 22. DNA sequence according to claim21, characterized in that the promoter corresponds to the sequence Seq.No.3 included between the nucleotides 907 and 990 of the sequence shownin FIG. 3, or a variant comprising the nucleotides 907 to 985, orcharacterized in that the promoter corresponds to any part of at leastten nucleotides of this sequence, naturally consecutive or not, capableof intervening in the control of the expression of a coding nucleotidesequence placed downstream in a bacterial cell host.
 23. Nucleotidesequence characterized in that it contains a region essentiallycomplementary to the 3′ end of the 16S RNA of a bacterial ribosome, saidsequence being capable of intervening at the post-transcriptional levelwhen the sequencing coding for a gene is being expressed, in particularwithout acting directly on the translation of the coding sequence to beexpressed, said nucleotide sequence being under the control of apromoter controlling said coding sequence.
 24. Nucleotide sequenceaccording to claim 23, characterized in that said sequence is isolatedfrom a bacterium, in particular from a Gram⁺ bacterium and moreparticularly from a bacterium of the Bacillus type.
 25. Nucleotidesequence according to claim 23, characterized in that said sequence isan S2 sequence containing a first region sufficiently complementary tothe 3′ end of the 16S RNA of a bacterial ribosome, in particular of aGram⁺ bacterium, more particularly of a bacterium of the Bacillus typeso that said first region is capable of hybridizing with a bacterialribosome or a part of a bacterial ribosome and a second regiondownstream from the first region, said second region containing asequence capable of having an additional positive effect at the level ofthe expression of the coding sequence.
 26. Nucleotide sequence accordingto claim 24, characterized in that the nucleotide sequence correspondsto the sequence Seq No.5 corresponding to the fragment comprising thenucleotides 1179 to 1556 of the sequence shown in FIG.
 3. 27. Nucleotidesequence according to claim 24, characterized in that the nucleotidesequence corresponds to the sequence Seq No.11 corresponding to thefragment comprising the nucleotides 1413 to 1556 of the sequence shownin FIG.
 3. 28. Nucleotide sequence according to claim 24, characterizedin that the nucleotide sequence corresponds to the sequence Seq No.8corresponding to the fragment comprising the nucleotides 1413 to 1461 ofthe sequence shown in FIG.
 3. 29. Nucleotide sequence according to claim24, characterized in that the nucleotide sequence or the downstreamregion corresponds to the sequence Seq No.9 corresponding to thefollowing DNA fragment: 5′-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3′3′-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′


30. Nucleotide sequence according to claim 24, characterized in that thenucleotide sequence or the downstream region corresponds to the sequenceSeq No.10 corresponding to the following DNA fragment:5′-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3′3′-GAACTTTCCTCCCTACGGATTTTTGCTTCTTG-5′


31. Nucleotide sequence according to claim 24, characterized in that thenucleotide sequence corresponds to the following DNA fragment:5′-GAAAGGAGG-3′ 3′-CTTTCCTCC-5′


32. Recombinant DNA sequence, characterized in that it comprises adefined coding sequence under the control of a DNA sequence according toany one of the claims 1 to
 22. 33. Expression vector characterized inthat it is modified at one of its sites by a DNA sequence according toany one of the claims 1 to 22, such that said DNA sequence intervenes inthe control of the expression of defined nucleotide coding sequence. 34.Expression vector according to claim 32, characterized in that it is aplasmid, for example an integrative plasmid or a replicative plasmid.35. Expression vector according to claim 34, characterized in that it isthe plasmid pHT7902′lacZ deposited with the CNCM on Apr. 20, 1993 underthe No. I-1301.
 36. Recombinant DNA sequence according to claim 32 orexpression vector according to claim 33 or 34, characterized in that thecoding nucleotide sequence which it contains is a toxic sequence towardsinsects and in particular a sequence with larvicidal activity, forexample the sequence coding for the cryIIIA gene of B. thuringiensis.37. Recombinant DNA sequence according to claim 32 or expression vectoraccording to claim 33 or 34, characterized in that the nucleotide codingsequence which it contains is a sequence coding for an enzyme. 38.Recombinant DNA sequence according to claim 32 or expression vectoraccording to claim 33 or 34, characterized in that the nucleotide codingsequence which it contains is a sequence coding for an antigen. 39.Recombinant cell host, characterized in that it is modified by a DNAsequence according to any one of the claims 1 to 32 or by an expressionvector according to any one of the claims 33 or
 34. 40. Cell hostaccording to claim 39, characterized in that it is a Bacillus, forexample B. thuringiensis or B. subtilis.
 41. Cell host according toclaim 40, characterized in that it is an asporogenic strain of Bacillus,for sample an asporogenic strain of B. subtilis in particular a straincapable of expressing the coding sequence of the DNA sequence accordingto any one of the claims 14 to 18 during the stationary phase of growth.42. Cell host, characterized in that it is the strain 407-OA::Km^(R)(pHT305D) deposited with the CNCM on May 3, 1994 under No. I-1412. 43.Production process for a recombinant protein encoded in a definednucleotide sequence, said process being characterized in that itcomprises: the introduction into a cell host of a vector according toany one of the claims 33 to 38, the growth of said cell host underconditions permitting the expression of said defined nucleotidesequence, and the recovery of the recombinant protein.